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269. Control of glucocorticoid responses in somatic cell hybrids between rat hepatoma cells and mouse tibroblasts
360
Abstracts
(5.5 x IO-“M) in cells from a premature infant dying of the Respiratory Distress Syndrome, the clinical expression of surfactant deficiency. In contrast, the initially higher incorporation of the precursors into lecithin by cells from a term infant without respiratory dysfunction was not increased by cortisol. Since these cell types behave metabolically in an “expected” way. we suggest that such preparations could play a useful role in studies of perinatal hormonal adaption.
269. Control of glucocorticoid responses in somatic cell hybrids between rat hepatoma cells and mouse tibroblasts LIPPMAN, M. E. and THOMPSON, E. B., National Cancer Institute, Bethesda. Md. 20014. U.S.A. We have prepared somatic cell hybrids between hepatoma tissue culture (HTC) and mouse fibroblast (L) cells. HTC cells respond to glucocorticoids by the induction of tyrosine aminotransferase and a specific t-RNA. L cells show a constellation of inhibitory responses to glucocorticoids. The hybrid cell (HL5) responds to steroids in a manner similar to the L cell parent alone. Heat inactivation studies of cell extracts suggest that the cytoplasmic glucocorticoid receptors (CR) from HTC and L cells are different and that the hybrid ceil contains both. An in vitro binding assay of CR to target nuclei was developed. Nuclei from HTC, L and HL5 hybrids can each distinguish CR from HTC or L cells. A cloned line of steroid unresponsive L cells which lacks cytoplasmic glucocorticoid receptor was developed and was hybridized with HTC cells. This hybrid, which contained CR. presumably from the HTC parent alone, showed neither HTC or L cell phenotypic responses to steroids. Thus HTC cell SBP could not repair the L cell receptor defect in the hybrid cell. This hybrid, which lacks the L-cell inhibitory responses shows that failure to induce the HTC aminotransferase is not due to the dominance of glucocorticoid provoked inhibitory responses. These results suggest that specificity of response to glucocorticoids could be mediated by different cytoplasmic receptors capable of combining with different nuclear sites.
270. Isolation and characterization of stable clonal lines of HTC (Hepatoma) cells, non-inducible for tyrosine aminotransferase by glucocorticoids THOMPSON. E. B., AVIV, D. and LIPPMAN, M. E., Laboratory of Biochemistry, National Cancer Institute, Bethesda, Md. 20853 U.S.A. HTC cells are a well-established rat hepatoma tissue culture line in which glucocorticoids induce tyrosine aminotransferase. By cloning the wild-type cells we have shown that clones with a wide range of inducibility can be isolated. We have now, through serial re-clonings, isolated a number of lines which inducelittle or not at all. They maintain this character stably through many subculturings. All the non-inducing lines contain basal levels of tyrosine aminotransferase and thus show that they are able to express the gene for the enzyme. They grow vigorously at a rate similar to that of uncloned cells. Chromosomal analysis shows that some ot the non-inducers contain double the modal number found in wild-type. The non-inducing lines all contain specific cytoplasmic steroid receptor sites at levels similar to those of wild-type or high-inducing clones. The receptors of the non-inducers have steroid affinities also quite similar to
inducible cells. It1 vitro nuclear binding activated steroid receptor complexes by non-inducible cells. These cells therefore induce because of some step subsequent receptor binding and activation. and uptake.
shows uptake of the nuclei of the appear to fail to to steroid uptake, possibly. nuclear
271. Influence of corticosteroids on phagocytosis in the lung SCHOKN, H. and LAFUMA, J.-C.E.A.-SPTE/ EURATOM-92260 Fontenay aux Roses, France The clearancerate of inhaled 59Fe0, particles in rats was investigated. Elimination of the stress by adaption of the animals to the inhalation procedure resulted in an increased clearance rate. Further improvement of the elimination of 59Fe-haematite was observed by suppression of corticosteroidbiosynthesiswith metopirone. Application ofcorticosterone and cortisol depressed the activity of phagocytosis, depending on dose and time of application. The percentage of radioactivity and the number of alveolar macrophages recovered by lung washing were reduced in relation to the decrease of the clearance rate. This would indicate an effect of the adrenal horfnones on the mobilization of the macrophages. As different synthetic glucocorticosteroids tested did not significantly influence these parameters (clearance rate, number of macrophages and radioactivity eluted), it would appear that this depressing effect on phagocytosis does not parallel its glucocosteroid potency. Some structureactivity relationships of corticosteroid action on alveolar macrophages could be postulated and possible applications in the treatment of inhalation accidents with toxic or radioactive substances are suggested. 272. Effect of corticosteroids on flavin nucleotide synthesis in rat tissues FAZEKAS, A. G. and SANDOR, T.. Dtpartement de MCdecine, UniversitC de Montreal, Montrkal, Qukbec. Canada Earlier investigations indicated that flavoprotein enzymes are involved in the mechanism of action of corticosteroids (aldosterone, cortisol). Since the formation and activity of flavoproteins is largely dependent on the availability of their coenzymes, the biosynthesis of flavin-adenine-dinucleotide (FAD) and flavin-mononucleotide (FMN) was investigated in the liver and kidney of intact, adrenalectomized (AX). AX-aldosterone treated and AX-corticosterone treated rats. Animals were killed 1 h following the injection of (2.‘V’) riboflavin (RF) and the radioactive flavins determined in their organs by a new method utilizing reverse isotope dilution and ion exchange chromatography. It was found that 3 days after adrenalectomy the biosynthesis of FAD in the liver decreased to 61”;, of control values of intact rats (100”;). Treatment of AX animals with aldosterone (10 pg/day for 3 days) increased FAD formation in the liver to 82”; of control values while FMN biosynthesis was not affected at all by these manipulations. AX animals treated with corticosterone or cortisol showed a marked decrease of hepatic FAD and FMN formation. None of the above changes could be observed in the kidneys. Studies irr ~~iftw using liver slices incubated with (2-lSC)RF and corticosterone yielded similar results. Stimulation of hepatic FAD-pyrophosphorylase by aldosterone indicates an extrarenal effect, antagonized by corticosterone and might be related to flavoprotein dependent oxydative processes enhanced by this steroid.
Abstracts
(5.5 x IO-“M) in cells from a premature infant dying of the Respiratory Distress Syndrome, the clinical expression of surfactant deficiency. In contrast, the initially higher incorporation of the precursors into lecithin by cells from a term infant without respiratory dysfunction was not increased by cortisol. Since these cell types behave metabolically in an “expected” way. we suggest that such preparations could play a useful role in studies of perinatal hormonal adaption.
269. Control of glucocorticoid responses in somatic cell hybrids between rat hepatoma cells and mouse tibroblasts LIPPMAN, M. E. and THOMPSON, E. B., National Cancer Institute, Bethesda. Md. 20014. U.S.A. We have prepared somatic cell hybrids between hepatoma tissue culture (HTC) and mouse fibroblast (L) cells. HTC cells respond to glucocorticoids by the induction of tyrosine aminotransferase and a specific t-RNA. L cells show a constellation of inhibitory responses to glucocorticoids. The hybrid cell (HL5) responds to steroids in a manner similar to the L cell parent alone. Heat inactivation studies of cell extracts suggest that the cytoplasmic glucocorticoid receptors (CR) from HTC and L cells are different and that the hybrid ceil contains both. An in vitro binding assay of CR to target nuclei was developed. Nuclei from HTC, L and HL5 hybrids can each distinguish CR from HTC or L cells. A cloned line of steroid unresponsive L cells which lacks cytoplasmic glucocorticoid receptor was developed and was hybridized with HTC cells. This hybrid, which contained CR. presumably from the HTC parent alone, showed neither HTC or L cell phenotypic responses to steroids. Thus HTC cell SBP could not repair the L cell receptor defect in the hybrid cell. This hybrid, which lacks the L-cell inhibitory responses shows that failure to induce the HTC aminotransferase is not due to the dominance of glucocorticoid provoked inhibitory responses. These results suggest that specificity of response to glucocorticoids could be mediated by different cytoplasmic receptors capable of combining with different nuclear sites.
270. Isolation and characterization of stable clonal lines of HTC (Hepatoma) cells, non-inducible for tyrosine aminotransferase by glucocorticoids THOMPSON. E. B., AVIV, D. and LIPPMAN, M. E., Laboratory of Biochemistry, National Cancer Institute, Bethesda, Md. 20853 U.S.A. HTC cells are a well-established rat hepatoma tissue culture line in which glucocorticoids induce tyrosine aminotransferase. By cloning the wild-type cells we have shown that clones with a wide range of inducibility can be isolated. We have now, through serial re-clonings, isolated a number of lines which inducelittle or not at all. They maintain this character stably through many subculturings. All the non-inducing lines contain basal levels of tyrosine aminotransferase and thus show that they are able to express the gene for the enzyme. They grow vigorously at a rate similar to that of uncloned cells. Chromosomal analysis shows that some ot the non-inducers contain double the modal number found in wild-type. The non-inducing lines all contain specific cytoplasmic steroid receptor sites at levels similar to those of wild-type or high-inducing clones. The receptors of the non-inducers have steroid affinities also quite similar to
inducible cells. It1 vitro nuclear binding activated steroid receptor complexes by non-inducible cells. These cells therefore induce because of some step subsequent receptor binding and activation. and uptake.
shows uptake of the nuclei of the appear to fail to to steroid uptake, possibly. nuclear
271. Influence of corticosteroids on phagocytosis in the lung SCHOKN, H. and LAFUMA, J.-C.E.A.-SPTE/ EURATOM-92260 Fontenay aux Roses, France The clearancerate of inhaled 59Fe0, particles in rats was investigated. Elimination of the stress by adaption of the animals to the inhalation procedure resulted in an increased clearance rate. Further improvement of the elimination of 59Fe-haematite was observed by suppression of corticosteroidbiosynthesiswith metopirone. Application ofcorticosterone and cortisol depressed the activity of phagocytosis, depending on dose and time of application. The percentage of radioactivity and the number of alveolar macrophages recovered by lung washing were reduced in relation to the decrease of the clearance rate. This would indicate an effect of the adrenal horfnones on the mobilization of the macrophages. As different synthetic glucocorticosteroids tested did not significantly influence these parameters (clearance rate, number of macrophages and radioactivity eluted), it would appear that this depressing effect on phagocytosis does not parallel its glucocosteroid potency. Some structureactivity relationships of corticosteroid action on alveolar macrophages could be postulated and possible applications in the treatment of inhalation accidents with toxic or radioactive substances are suggested. 272. Effect of corticosteroids on flavin nucleotide synthesis in rat tissues FAZEKAS, A. G. and SANDOR, T.. Dtpartement de MCdecine, UniversitC de Montreal, Montrkal, Qukbec. Canada Earlier investigations indicated that flavoprotein enzymes are involved in the mechanism of action of corticosteroids (aldosterone, cortisol). Since the formation and activity of flavoproteins is largely dependent on the availability of their coenzymes, the biosynthesis of flavin-adenine-dinucleotide (FAD) and flavin-mononucleotide (FMN) was investigated in the liver and kidney of intact, adrenalectomized (AX). AX-aldosterone treated and AX-corticosterone treated rats. Animals were killed 1 h following the injection of (2.‘V’) riboflavin (RF) and the radioactive flavins determined in their organs by a new method utilizing reverse isotope dilution and ion exchange chromatography. It was found that 3 days after adrenalectomy the biosynthesis of FAD in the liver decreased to 61”;, of control values of intact rats (100”;). Treatment of AX animals with aldosterone (10 pg/day for 3 days) increased FAD formation in the liver to 82”; of control values while FMN biosynthesis was not affected at all by these manipulations. AX animals treated with corticosterone or cortisol showed a marked decrease of hepatic FAD and FMN formation. None of the above changes could be observed in the kidneys. Studies irr ~~iftw using liver slices incubated with (2-lSC)RF and corticosterone yielded similar results. Stimulation of hepatic FAD-pyrophosphorylase by aldosterone indicates an extrarenal effect, antagonized by corticosterone and might be related to flavoprotein dependent oxydative processes enhanced by this steroid.